Maturation and function of cystic fibrosis transmembrane conductance regulator variants bearing mutations in putative nucleotide-binding domains 1 and 2.

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Abstract
One feature of the mutations thus far found to be associated with the disease cystic fibrosis (CF) is that many of them are clustered within the first nucleotide-binding domain (NBD) of the CF transmembrane conductance regulator (CFTR). We sought to discover the molecular basis for this clustering by introducing into the two NBDs of CFTR mutations either mimicking amino acid changes associated with CF or altering residues within highly conserved motifs. Synthesis and maturation of the mutant CFTR were studied by transient expression in COS cells. The ability of the altered proteins to generate cyclic AMP-stimulated anion efflux was assessed by using 6-methoxy-N-(sulfopropyl) quinolinium (SPQ) fluorescence measurements in HeLa cells expressing mutated plasmids. The results show that (i) all CF-associated mutants, with one exception, lack functional activity as measured in the SPQ assay, (ii) mutations in NBD1 are more sensitive to the effects of the same amino acid change than are the corresponding mutations in NBD2, (iii) cells transfected with plasmids bearing CF-associated mutations commonly but not exclusively lack mature CFTR, (iv) NBD mutants lacking mature CFTR fail to activate Cl- channels, and (v) the glycosylation of CFTR, per se, is not required for CFTR function. We reason that the structure of NBD1 itself or of the surrounding domains renders it particularly sensitive to mutational changes. As a result, most NBD1 mutants, but only a few NBD2 mutants, fail to mature or lack functional activity. These findings are consistent with the observed uneven distribution of CFTR missense mutations between NBD1 and NBD2 of CF patients.